Linked Life Data

11679164

Source:http://linkedlifedata.com/resource/pubmed/id/11679164

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2001-10-26
pubmed:abstractText
DNA replication proteins represent a class of extremely well-established anti-infective drug targets for which improvements in assay technology are required in order to support enzyme characterization, HTS, and structure-activity relationship studies. Replication proteins are conventionally assayed using precipitation/filtration or gel-based techniques, and are not yet all suitable for conversion into homogeneous fluorescence-based formats. We have therefore developed radiometric assays for these enzymes based upon FlashPlate technology that can be applied to a wide range of targets using a common set of reagents. This approach has allowed the rapid characterization of DNA polymerase, DNA primase, and DNA helicase activities. The resultant 96-/384-well microplate assays are suitable for primary HTS, hit selectivity determination, and/or elucidating the mechanism of action of inhibitors. In all cases, biotinylated DNA oligonucleotide substrates were tethered to streptavidin-coated scintillant-embedded FlashPlate wells. Various adaptations were employed for each enzyme activity. For DNA polymerase, a short complementary oligonucleotide primer was annealed to the longer tethered oligonucleotide, and polymerization was measured by incorporation of [(3)H]-dNTPs onto the growing primer 3' end. For DNA primase, direct synthesis of short oligoribonucleotides complementary to the tethered DNA strand was measured by incorporation of [(3)H]-rNTPs or by subsequent polymerase extension with [(3)H]-dNTPs from unlabeled primers. For DNA helicase, unwinding of a [(33)P]-labeled oligonucleotide complementary to the tethered oligonucleotide was measured. This robust and flexible system has a number of substantial advantages over conventional assay techniques for this difficult class of enzymes.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
1087-0571
pubmed:author
pubmed:issnType
Print
pubmed:volume
6
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
39-46
pubmed:dateRevised
2011-5-23
pubmed:meshHeading
pubmed:year
2001
pubmed:articleTitle
FlashPlate scintillation proximity assays for characterization and screening of DNA polymerase, primase, and helicase activities.
pubmed:affiliation
Molecular Interactions and New Assay Technologies, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Essex, UK.
pubmed:publicationType
Journal Article, Comparative Study